Bacillus thuringiensis is a microbial insecticide that is widely used to control insects, including mosquitoes and black flies. The long-range goal of this project is to investigate the binding and mechanism of action of several mosquitocidal proteins against key pestiferous mosquito species, Anopheles gambiae, Aedes aegypti and Culex quinquefaciatus. The main mosquitocidal toxins of interest are the toxins of B. thuringiensis var. israelensis (Bti), Cry4Aa, Cry4Ab and Cry11Aa. Other mosquitocidal toxins, Cry11Ba, Cry19Aa and Cry2Aa, will also be investigated. The hypothesis to be tested is that these toxins bind to specific receptors on the mosquito midgut as recognized in model insect-toxin studies (the Lepidoptera-toxin paradigm); i.e., an array of mosquito midgut proteins (cadherin-like and aminopeptidases) and glycoproteins bind the toxins; and, that domains II and Ill of the are the interacting binding epitopes. A corollary hypothesis is that the reduced ability of mosquitoes to develop resistance to Bti is due to a combination of toxins (Cry4Aa, Cry4Ab and Cry11Aa) each of which binds to a unique receptor or non-competing binding site. The specific aims of the proposal are: (1) Test the competition, saturation and irreversible binding of Cry4Aa, 4Ba, 11Aa, 11Ba, 19Aa and 2Aa toxins to mosquito BBMV and purified receptors. New mosquitocidal activity has been introduced into Cry4Ba (Culex activity) and 19Aa (Aedes activity). The mechanistic basis for these new activities will be investigated. (2) Examine the mechanism of action of mosquitocidal of these toxins, in comparison to the Lepidoptera toxins paradigm; specifically, to define the binding epitopes of these toxins to brush border membrane vesicles (BBMV) of the mosquitoes Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. (3) Examine the nature of mosquito midgut receptors in relation to what has been learned from Lepidoptera receptor paradigm; specifically, to identify the specific binding of these mosquitocidal Cry proteins on aminopeptidases, cadherin-like proteins, glycoproteins and other potential receptors of these mosquitoes.